The overall goal of this protocol is to deliver Neurotrophin 4 for the induction of aging rat laryngeal muscle remodeling. This method can help answer key questions related to laryngeal muscle function. For example, can age related muscle atrophy be reversed to improve vocal function.
The main advantage of this technique is that it can be easily translated to human models of vocal aging. We can use injection methods that are typically used by otolaryngologists in humans to inject the solutions that are necessary to improve vocal function. Generally individuals new to this method will struggle because of the delicacy of the implantation and injection procedures.
Demonstrating the procedure will be Tanya Seward, a technician from the department of physiology and Richard Andreatta, an associate professor in communication sciences and disorders, both of the University of Kentucky. To implant the pump, first place the rat in the ventral position in an aseptic surgical area. Wet the back and neck with iodine.
Then scrub the dorsal aspect of the neck with 70%ethanol. Next move the animal onto a 34 degree Celsius heating pad and administer pre-anesthetic medication. Fill the aseptically prepared osmotic pumps with 50 microliters of Neurotrophin 4.
And use a scalpel to make a two centimeter wide, horizontal incision through the skin just posterior to the space between the scapulae. Life the posterior edge of the incision with forceps and gently insert the tips of the hemostat posterior to the incision. When the tip is approximately two centimeters posterior to the incision, open the handles on the hemostat to make a hollow pocket subcutaneous to the incision site.
Insert the pump into the pocket, orienting the end of the delivery portal such that any other interaction between the treatment and the healing of the pocket incision site is minimized. Then, use a 5-0 nylon suture thread, hemostat and forceps to close the pump placement incision and monitor the animal until it is fully alert and ambulatory. 30 minutes before the inject, warm aseptically prepared 50 microliter doses of the Neurotrophin or saline in a 25 degree Celsius water bath.
When the treatments are ready, recline an anesthetized rat in the supine position on a plexiglass platform and use the guide wire to suspend the animal via the top frontal incisors. Next, attach a 30 gauge, 50 millimeter, 100 microliter syringe to a 1.9 millimeter 30 degrees sinus endoscope. And attach the syringe assembly to a jig on the outer wall of the endoscope.
When the syringe is secure, load the treatment and attach the endoscope assembly to the camera. Then, use rubber tipped forceps to extend the tongue to the side and insert a plastic speculum to maintain oral patency. And turn on the halogen light source to the endoscope.
With the video recorder on, immerse the distal end of the endoscope in warm water for a few seconds to minimize the development of condensation on the glass tip. And carefully insert the needle into the area of the left vocal fold, using visual feedback from the monitor as a guide. When the vocal fold is fully visible, inject the needle into the left thyroid arytenoid lateral to the white, medial edge of the vocal fold.
And depress the plunger to deliver the treatment. If the epiglottis is stuck closed, preventing a clear view of the larynx, use an upward sweeping motion with the syringe tip to gently unstick the tissue. Then withdraw the needle and place the animal in its home cage on a heating pad until full recumbency.
Typically, a change in fiber size is observed with age in Neurotrophin 4 treated animals. A lower lever of fibrosis and fiber generation is also observed in aged Neurotrophin treated animals. While the quantity of neural muscular junctions is increased.
Once mastered, the osmotic pump and the injection procedure, can each be completed in one hour if they're performed properly. While attempting this procedure, it's important to remember to use a slow and gentle with the needle to prevent scratching and injury around the laryngeal area. Though this method can provide insight into the mechanisms of vocal fold function during the aging process, it can also be applied to other systems such as the pharynx and swallowing related disorders.
